Stable formulations of lipid-encapsulated rna

Abstract

The present disclosure provides lipid nanoparticle (LNP)-encapsulated RNA compositions that are stable without sub-zero storage. As described in detail herein, the compositions comprise an LNP-encapsulated RNA and a combination of excipients that stabilize the composition for at least three months or at least six months under refrigerated conditions.

1 . A lipid nanoparticle (LNP)-encapsulated RNA composition, comprising an LNP-encapsulated RNA and a combination of excipients providing stability for at least three months with refrigeration in the range of 2° C.-8° C. 
     
     
         2 . The composition of  claim 1 , wherein the composition is stable for at least three months at 2° C. 
     
     
         3 . The composition of  claim 1 , wherein the composition is stable for at least six months at 2° C. 
     
     
         4 . The composition of  claim 1 , wherein the composition is stable for at least three months at 4° C. 
     
     
         5 . The composition of  claim 1 , wherein the composition is stable for at least six months at 4° C. 
     
     
         6 . The composition of  claim 1 , wherein the composition is stable for at least three months at 8° C. 
     
     
         7 . The composition of  claim 1 , wherein the composition is stable for at least six months with refrigeration in the range of 2° C.-8° C. 
     
     
         8 . The composition of  claim 1 , wherein at least 60% of the RNA remains intact after 6 months at 2-8° C., as compared to the same composition at time zero, optionally wherein at least 70% of the RNA remains intact after 6 months at 2-8° C., as compared to the same composition at time zero, optionally wherein at least 80% of the RNA remains intact after 6 months at 2-8° C., as compared to the same composition at time zero, optionally wherein at least 90% of the RNA remains intact after 6 months at 2-8° C., as compared to the same composition at time zero. 
     
     
         9 . The composition of any one of  claims 1 to 8 , wherein the excipients comprise an antioxidant, a non-ionic surfactant, a stabilizing agent, and a pH buffer. 
     
     
         10 . The composition of  claim 9 , wherein the antioxidant is selected from one or more of methionine, cysteine, and ascorbic acid. 
     
     
         11 . The composition of  claim 10 , wherein the antioxidant is methionine or cysteine. 
     
     
         12 . The composition of  claim 11 , wherein the antioxidant is methionine, and the concentration of methionine is optionally from about 0.05% to about 1.50% w/v, or from about 0.05% to about 0.5% w/v, or from or about 0.05% to about 0.25% w/v, or from about 
     
     
       0. 05% to about 0.15% w/v. 
     
     
         13 . The composition of any one of  claims 9 to 12 , wherein the non-ionic surfactant is a polysorbate, optionally polysorbate-20. 
     
     
         14 . The composition of  claim 13 , wherein the composition comprises a polysorbate at a concentration of about 0.001% to about 0.1% w/v, or from about 0.005% to about 0.05% w/v, or about 0.01% w/v, and which is optionally polysorbate-20. 
     
     
         15 . The composition of any one of  claims 9 to 12 , wherein the non-ionic surfactant is a poloxamer, and optionally Poloxamer 188. 
     
     
         16 . The composition of  claim 15 , wherein the composition comprises the poloxamer at a concentration of about 0.001% to about 0.1% w/v, or from about 0.005% to about 0.05% w/v, or about 0.01% w/v. 
     
     
         17 . The composition of any one of  claims 9 to 16 , wherein the stabilizing agent is selected from one or more of glycine, sorbitol, and gelatin. 
     
     
         18 . The composition of  claim 17 , wherein the stabilizing agent is glycine. 
     
     
         19 . The composition of  claim 18 , wherein the concentration of glycine is from about 0.25% to about 15% w/v, or from about 0.5% to about 15% w/v, or from about 0.25% to about 10% w/v, or from about 0.25% to about 5% w/v, or from about 0.5% to about 2.5% w/v, or about 1.5% w/v. 
     
     
         20 . The composition of  claim 19 , comprising or consisting essentially of the following excipients in buffered solution:
 methionine from about 0.05% to about 1.50% w/v;   polysorbate-20 from about 0.001% to about 0.1% w/v; and   glycine from about 0.25% to about 15% w/v.   
     
     
         21 . The composition of  claim 20 , comprising or consisting essentially of the following excipients in buffered solution:
 methionine from about 0.05 to about 0.25% w/v;   polysorbate-20 from about 0.005% to about 0.05% w/v; and   glycine from about 0.5% to about 2.5% w/v.   
     
     
         22 . The composition of  claim 21 , comprising or consisting essentially of the following excipients in buffered solution: methionine at about 0.075 to about 0.15% w/v, polysorbate-20 at about 0.01% w/v, and glycine at about 1.5% w/v. 
     
     
         23 . The composition of any one of  claims 1 to 22 , wherein the composition is pH buffered in the range of about 6.0 to about 8.0. 
     
     
         24 . The composition of  claim 23 , wherein the composition is buffered at about pH 7.4. 
     
     
         25 . The composition of  claim 23 or 24 , wherein the pH buffer is a phosphate buffer. 
     
     
         26 . The composition of  claim 23 or 24 , wherein the pH buffer is selected from a Tris-EDTA buffer, a histidine buffer, and a sodium citrate buffer. 
     
     
         27 . The composition of  claim 26 , wherein the pH buffer is Tris-EDTA buffer, optionally wherein the Tris-EDTA buffer is at a concentration of 1× to 10×, optionally wherein the Tris-EDTA buffer is at a concentration of 1× to 5×, optionally wherein the Tris-EDTA buffer is at a concentration of 1× to 3×, optionally wherein the Tris-EDTA buffer is a concentration of 1×. 
     
     
         28 . The composition of any one of  claims 1 to 27 , wherein the composition comprises or further comprises a metal ion chelator. 
     
     
         29 . The composition of  claim 28 , wherein the metal ion chelator is EDTA or salt thereof. 
     
     
         30 . The composition of  claim 29 , wherein the metal ion chelator is disodium EDTA. 
     
     
         31 . The composition of  claim 29 or 30 , wherein the concentration or EDTA or disodium EDTA is from about 0.01 mM to about 1 mM, or from about 0.05 mM to about 0.5 mM, or about 0.1 mM. 
     
     
         32 . The composition of any one of  claims 1 to 31 , wherein the composition further comprises an excipient that reduces exposure of the RNA to water. 
     
     
         33 . The composition of  claim 32 , wherein the excipient that reduces exposure of the mRNA to water is sucrose. 
     
     
         34 . The composition of any one of  claims 1 to 33 , wherein the composition further comprises an excipient that reduces degradation of the RNA by free-radical oxidation. 
     
     
         35 . The composition of  claim 34 , wherein the excipient that reduces degradation of the RNA by free-radical oxidation is one or more of ethanol and histidine. 
     
     
         36 . The composition of  claim 35 , wherein ethanol is included as an excipient at 200 mM or less, or about 150 mM or less, or about 100 mM of less, or about 50 mM or less. 
     
     
         37 . The composition of any one of  claims 34 to 36 , wherein the composition comprises histidine at a concentration of from about 0.01% w/v to about 1% w/v, or from about 0.05% w/v to about 0.5% w/v, or about 0.1% w/v. 
     
     
         38 . The composition of any of  claims 1 to 8 , wherein the excipients comprise one or more salts, and a pH buffer. 
     
     
         39 . The composition of  claim 38 , further comprising one or more additional excipients selected from a non-ionic surfactant, antioxidants, stabilizing agents, and cryoprotectants. 
     
     
         40 . The composition of  claim 38 or 39 , wherein the one or more salts are selected from the group of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. 
     
     
         41 . The composition of  claim 40 , wherein the one or more salts comprise at least one chloride salt and at least one sulfate salt; and/or wherein the one or more salts comprise at least one potassium salt and at least one sodium salt. 
     
     
         42 . The composition of  claim 40 , wherein the one or more salts comprise or consist essentially of:
 (a) sodium sulfate, potassium chloride, and sodium chloride;   (b) sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate;   (c) potassium sulfate, sodium chloride, and potassium chloride;   (d) sodium chloride, sodium sulfate, and potassium sulfate;   (e) sodium chloride and potassium sulfate;   (f) sodium chloride and potassium chloride;   (g) potassium chloride, potassium sulfate, and sodium sulfate;   (h) potassium chloride and potassium sulfate;   (i) sodium sulfate and sodium chloride;   (j) potassium chloride and sodium sulfate;   (k) potassium sulfate and sodium sulfate;   (l) sodium chloride;   (m) potassium chloride;   (n) sodium sulfate; or   (o) potassium sulfate.   
     
     
         43 . The composition of  claim 40 , wherein the one or more salts comprise or consist essentially of potassium sulfate, sodium chloride, and potassium chloride. 
     
     
         44 . The composition of  claim 40 , wherein the one or more salts comprise or consist essentially of sodium sulfate and sodium chloride. 
     
     
         45 . The composition of any one of  claims 40 to 44 , wherein the concentration of one or more sulfate salts is from about 2.5 mM to about 150 mM, or from about 10 mM to about 75 mM, or from about 25 mM to about 75 mM, or from about 33 mM to about 75 mM. 
     
     
         46 . The composition of any of  claims 40 to 44 , wherein the concentration of one or more chloride salts is from about 10 mM to about 1,000 mM, or from about 50 mM to about 600 mM, or from about 100 mM to about 500 mM, or is from about 100 mM to about 200 mM, or is from about 136 mM to about 200 mM. 
     
     
         47 . The composition of  claim 45 or 46 , wherein the concentration of potassium sulfate is about 43 mM, the concentration of sodium sulfate is about 34 mM, the concentration of sodium chloride is about 136 mM, and the concentration of potassium chloride is about 136 mM. 
     
     
         48 . The composition of  claim 45 or 46 , wherein the concentration of potassium sulfate is about 75 mM, the concentration of sodium sulfate is about 75 mM, the concentration of sodium chloride is about 200 mM, and the concentration of potassium chloride is about 200 mM. 
     
     
         49 . The composition of  claim 45 or 46 , wherein the concentration of potassium sulfate is about 33 mM, the concentration of potassium chloride is about 199 mM, and the concentration of sodium chloride is about 190 mM. 
     
     
         50 . The composition of  claim 45 or 46 , wherein the concentration of potassium sulfate is about 47 mM, the concentration of potassium chloride is about 180 mM, and the concentration of sodium chloride is about 141 mM. 
     
     
         51 . The composition of  claim 45 or 46 , wherein the concentration of sodium sulfate is about 48 mM and the concentration of sodium chloride is about 192 mM. 
     
     
         52 . The composition of  claim 45 or 46 , wherein the concentration of the sodium sulfate is about 75 mM, the concentration of potassium chloride is about 125 mM, and the concentration of sodium chloride is about 200 mM. 
     
     
         53 . The composition of any of  claims 39 to 52 , wherein the composition comprises a nonionic surfactant, optionally wherein the nonionic surfactant is selected from polysorbate-20, polysorbate-60, and polysorbate-80. 
     
     
         54 . The composition of  claim 53 , wherein the nonionic surfactant is in a concentration of about 0.005% to about 0.04%, optionally wherein the concentration is about 0.01% w/v, and the nonionic surfactant is optionally polysorbate-20. 
     
     
         55 . The composition of any of  claims 38 to 54 , wherein the pH buffer is selected from Tris-EDTA (TE), PBS, histidine, Tris acetate, and sodium citrate. 
     
     
         56 . The composition of  claim 55 , wherein the pH buffer is Tris-EDTA (TE) buffer, optionally wherein the Tris-EDTA (TE) buffer is at a concentration of 1× to 10×, optionally wherein the Tris-EDTA (TE) buffer is at a concentration of 1× to 5×, optionally wherein the Tris-EDTA (TE) buffer is at a concentration of 1× to 3×, optionally wherein the Tris-EDTA (TE) buffer is a concentration of 1×. 
     
     
         57 . The composition of any of  claims 39 to 56 , wherein the composition comprises one or more antioxidants, optionally wherein the one or more antioxidants is selected from methionine and cysteine. 
     
     
         58 . The composition of  claim 57 , wherein the one or more antioxidants comprises cysteine, optionally wherein the cysteine is in a concentration of about 0.05% to about 0.5%, or from about 0.075% to about 0.5%, or from about 0.10% to about 0.5%, or from about 0.10% to about 0.20%, or about 0.10% to about 0.15% w/v. 
     
     
         59 . The composition of  claim 57 or 58 , wherein the one or more antioxidants comprises methionine, optionally wherein the methionine is in a concentration of from about 0.025% to about 3.0% w/v, or from about 0.025% to about 0.5% w/v, or from about 0.075% to about 0.5%, or from about 0.05 to about 0.25% w/v, or about 0.15% w/v. 
     
     
         60 . The composition of any one of  claims 39 to 59 , wherein the composition comprises a cryoprotectant, optionally wherein the cryoprotectant is sucrose, optionally wherein the sucrose is in a concentration of about 5% to about 20%, optionally wherein the sucrose is in a concentration from about 10% to about 15% w/v, optionally wherein the sucrose is in a concentration of about 10%. 
     
     
         61 . The composition of any one of  claims 39 to 60 , wherein the composition comprises a stabilizing agent, optionally wherein the stabilizing agent is selected from one or more of glycine, sorbitol, and gelatin. 
     
     
         62 . The composition of any claim  62  wherein the stabilizing agent is glycine. 
     
     
         63 . The composition of  claim 61 or 62  wherein the stabilizing agent is at a concentration of about 0.05% to about 15% w/v, optionally wherein the stabilizing agent is at a concentration of about 0.5% to about 10% w/v, optionally wherein the stabilizing agent is at a concentration of about 1% to about 2%, optionally wherein the stabilizing agent is at a concentration of about 1.5%. 
     
     
         64 . The composition of any one of  claims 1 to 63 , wherein the RNA is messenger RNA (mRNA). 
     
     
         65 . The composition of  claim 64 , wherein the RNA is an mRNA encoding a component of an infectious agent. 
     
     
         66 . The composition of  claim 65 , wherein the infectious agent is a virus. 
     
     
         67 . The composition of  claim 66 , wherein the virus is a coronavirus, such as a betacoronavirus. 
     
     
         68 . The composition of  claim 67 , wherein the betacoronavirus is SARS-COV. 
     
     
         69 . The composition of any one of  claims 66 to 68 , wherein the mRNA encodes one or more viral structural proteins or one or more polypeptides derived from virus proteins. 
     
     
         70 . The composition of  claim 69 , wherein the mRNA encodes a coronavirus Spike protein(S). 
     
     
         71 . The composition of  claim 69 or claim 70 , wherein the mRNA encodes M (membrane) glycoprotein, E (envelope) protein, and/or N (nucleocapsid) protein. 
     
     
         72 . The composition of  claim 66 , wherein the mRNA encodes one or more influenza proteins, such as neuraminidase (NA), hemagglutinin (HA), matrix protein 2 (M2), and/or nucleoprotein (NP). 
     
     
         73 . The composition of  claim 64 , wherein the mRNA encodes a therapeutic protein. 
     
     
         74 . The composition of  claim 66 , wherein the RNA encodes a varicella zoster protein, optionally wherein the varicella zoster protein is selected from glycoprotein E and glycoprotein I. 
     
     
         75 . The composition of any one of  claims 64 to 74 , wherein the composition is injectable. 
     
     
         76 . A method for expressing an mRNA in cells of a patient, comprising, administering the composition of any one of  claims 1 to 75  in said patient. 
     
     
         77 . A method for expressing an mRNA in cells of a mammal, comprising, administering the composition of any one of  claims 1 to 75  in said mammal, optionally wherein the mammal is selected from the group consisting of a pig and a cow. 
     
     
         78 . A method for expressing an mRNA in cells of a bird, comprising. administering the composition of any one of  claims 1 to 75  in said bird, optionally wherein the bird is a chicken. 
     
     
         79 . A method for increasing the stability of an RNA by formulating the RNA in a composition of any of  claims 1 to 75 .